Cellular and plasma cholesterol levels are maintained through tightly controlled mechanisms, which regulate the expression and activity of key metabolic genes at both the transcriptional and post-transcriptional level. Alterations in the control of cholesterol homeostasis can lead to pathological processes, including atherosclerosis, the most common cause of mortality in Western societies (Lusis 2000, Glass and Witztum 2001). Epidemiological studies have identified many environmental and genetic factors that contribute to atherogenesis. In particular, high levels of low-density lipoprotein (LDL) cholesterol and low levels of high-density lipoprotein (HDL) cholesterol are associated with increased cardiovascular disease (CVD) risk (Lusis 2000, Glass and Witztum 2001). As a result, substantial therapeutic progress has resulted from the widespread use of statins (Gould, Rossouw et al. 1998) and other lipid-lowering drugs aimed at lowering plasma LDL-cholesterol (LDL-C). Despite this, statins are not sufficient to prevent the progression of atherosclerosis in many individuals and there is considerable evidence that quantitatively important determinants of disease susceptibility remain to be identified (Hennekens 1998, Sjouke, Kusters et al. 2011).
In humans, the majority of serum cholesterol is transported as cholesterol esters in LDL particles. To ensure that blood cholesterol levels are balanced, LDL is constantly internalized. The uptake of LDL and other ApoE/ApoB containing lipoproteins occurs through the LDL receptor (LDLR) and is a classic example of receptor-mediated endocytosis (Brown and Goldstein 1976, Brown and Goldstein 1986). The circulating level of LDL is determined in large part by its rate of uptake through this pathway, as evidenced by mutations in Ldlr or ApoB, which lead to the massive accumulation of LDL in patients with familial hypercholesterolemia (FH) (Brown and Goldstein 1974, Maxfield and Tabas 2005). The expression of the LDLR is tightly controlled by feedback mechanisms that operate at both transcriptional and post-transcriptional levels. One of the classical transcriptional regulators of the LDLR is the ER-bound sterol regulatory element-binding protein (SREBP). SREBPs are members of the basic helix-loop-helix leucine zipper (bHLH-Zip) family that bind to sterol response elements (SREs) and promote gene expression (Goldstein and Brown 1990, Brown and Goldstein 1997). In mammals there are three isoforms: SREBP1a and SREBP1c, encoded by the Srebp1 gene, and SREBP2, encoded by the Srebp2 gene. While SREBP1c is regulated by insulin and oxysterols and preferentially enhances the transcription of genes involved in fatty acid synthesis, SREBP2 is regulated by intracellular cholesterol concentrations and is the main regulator of de novo cholesterol biosynthesis (Goldstein and Brown 1990, Brown and Goldstein 1997). When intracellular levels of cholesterol are high, the ER-bound sterol regulatory element-binding proteins (SREBPs), such as SREBP2, coordinate the down-regulation of the LDLR, as well as 3-hydroxy-3methylglutaryl coenzyme A reductase (HMGCR), the rate-limiting enzyme of cholesterol biosynthesis (Goldstein and Brown 1990, Brown and Goldstein 1997). Conversely, when sterol concentrations are low, SREBPs, such as SREBP2, upregulate HMGCR and the LDLR, thereby enhancing LDL clearance from the plasma and ensuring that intracellular cholesterol levels are maintained (Goldstein and Brown 1990, Brown and Goldstein 1997). Additionally, the LDLR is also subject to post-transcriptional regulation such as its proprotein convertase sutilisin/kexin type 9 (PCSK9)-dependent degradation and inducible degrader of idol (IDOL)-dependent ubiquitination (Park, Moon et al. 2004, Zelcer, Hong et al. 2009)
While several key transcriptional regulators of cellular and systemic lipid levels have been identified, post-transcriptional mediators of cholesterol metabolism, including microRNAs, are less well-characterized and just beginning to emerge. MicroRNAs (miRNAs) are short (˜22 nt), evolutionary conserved, single-stranded RNAs that control the expression of complementary target mRNAs, leading to their transcript destabilization, translational inhibition, or both (Ambros 2004, Filipowicz, Bhattacharyya et al. 2008, Bartel 2009). As such, they are crucial for the development and maintenance of tissues, both in health and disease states. Recently, it has been suggested that miR-122, miR-33, miR-758, miR-106b, and miR-144 are involved in control of lipid metabolism (Krutzfeldt, Rajewsky et al. 2005, Esau, Davis et al. 2006, Najafi-Shoushtari, Kristo et al. 2010, Ramirez, Davalos et al. 2011, Rayner, Esau et al. 2011, Kim, Yoon et al. 2012, de Aguiar Vallim, Tarling et al. 2013, Ramirez, Rotllan et al. 2013). However, the effect of miRNAs on LDLR activity has not been described.
miRNAs typically control the expression of their target transcripts by binding to the 3′-UTR of mRNAs. In mammals, the most consistent requirement of miRNA:target interaction, although not always essential, is the contiguous and perfect base pairing of nucleotides 2-8 (the ‘seed’) at the 5′ end of the miRNA (Ambros 2004, Bartel 2004, Filipowicz, Bhattacharyya et al. 2008, Bartel 2009). Given the shortness of the seed region, it is no surprise that a single miRNA can potentially regulate hundreds of genes that are involved in multiple signaling cascades or cellular mechanisms (Bartel 2004). While these numbers emphasize the regulatory potential of miRNAs, they also reflect how difficult it is to determine the function of a given miRNA, as not all predicted targets will contribute to a phenotype. Ascertaining the biological function of miRNAs in regulating a physiological process, therefore, is complex and relies on systematic, unbiased experiments in living cells or organisms.